Summary The modification of intracellular proteins by O-linked ?-N-acetylglucosamine (O-GlcNAc) has emerged as a novel regulator of cytoprotection. Numerous forms of cellular injury, including cardiac ischemic preconditioning (acute, prolonged, and remote), lead to elevated levels of O-GlcNAc in both in vivo and in vitro models. Elevating O-GlcNAcylation before, or immediately after, the induction of cellular injury is protective in models of ischemia reperfusion (I/R) injury, as well as heat stress, oxidative stress, endoplasmic reticulum stress, hypoxia, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a novel endogenous cardioprotective agent. To date, the majority of work studying the O-GlcNAc modification in models of I/R injury has focused on identifying the proteins and mechanisms by which O-GlcNAc mediates cardioprotection. However, understanding the regulation of the O-GlcNAc modification during injury is critical and is yet unstudied. The goal of this proposal is to map the regulatory networks of the enzymes that cycle the O-GlcNAc modification, the O- GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Specifically, we will complete the following aims: Aim 1. Define the impact of protein-protein interactions on OGT and OGA activity and substrate targeting in the injured heart. Quantitative mass spectrometry will be used to identify protein-interactors of OGT and OGA. A combination of biochemical approaches will be used to interrogate the role of these effector- proteins on O-GlcNAc cycling in the ischemic heart. Aim 2. Map the OGT and OGA covalent-regulatory networks in the injured heart. The O-GlcNAc modification sites, and other covalent-regulators, of OGT and OGA will be identified. The impact of O- GlcNAcylation on OGT/OGA function will be assessed in models of oxidative stress and I/R injury. Collectively, we anticipate that these studies will define the pathways that regulate OGT, OGA, and O- GlcNAcylation during I/R injury. This critical insight will provide a framework for investigating novel therapeutic targets for myocardial infarction and an understanding about how the O-GlcNAc-mediated stress response is dysregulated contributing to cardiovascular disease